Cathepsin K deficiency partially inhibits, but does not prevent, bone destruction in human tumor necrosis factor-transgenic mice.

OBJECTIVE: Cathepsin K is believed to have an eminent role in the pathologic resorption of bone. However, several studies have shown that other proteinases also participate in this process. In order to clarify the contribution of cathepsin K to the destruction of arthritic bone, we applied the human tumor necrosis factor (hTNF)-transgenic mouse model, in which severe polyarthritis characterized by strong osteoclast-mediated bone destruction develops spontaneously. METHODS: Arthritis was evaluated in hTNF-transgenic mice that were either wild-type for cathepsin K (CK(+/+)), heterozygous for cathepsin K (CK(+/-)), or deficient in cathepsin K (CK(-/-)). Arthritic knee joints were prepared for standard histologic assessment aimed at establishing a semiquantitative score for joint destruction and quantification of the area of bone erosion. Additionally, microfocal computed tomography was performed to visualize bone destruction in mice with the different CK genotypes. CK(+/+) and CK(-/-) osteoclasts were generated in vitro, and their proteinase expression profiles were compared by complementary DNA array analysis, real-time polymerase chain reaction, and activity assays. RESULTS: Although the area of bone erosion was significantly reduced in hTNF-transgenic CK(-/-) mice, the absence of cathepsin K did not completely protect against inflammatory bone lesions. Several matrix metalloproteinases (MMPs) and cathepsins were expressed by in vitro-generated CK(-/-) osteoclasts, without marked differences from CK(+/+) osteoclasts. MMP activity was detected in CK(-/-) osteoclasts, and MMP-14 was localized by immunohistochemistry in inflammatory bone erosions in hTNF-transgenic CK(-/-) mice, suggesting MMPs as potential contributors to bone destruction. Additionally, we detected a reduction in osteoclast formation in cathepsin K-deficient mice, both in vitro and in vivo. CONCLUSION: The results of our experiments raise doubts about a crucial role of cathepsin K in arthritic bone destruction.

Cooperation of Ras- and c-Myc-dependent pathways in regulating the growth and invasiveness of synovial fibroblasts in rheumatoid arthritis.

OBJECTIVE: To study the specific contribution of MAP kinase activator c-Raf-1 and one of its downstream transcription factors, c-Myc, to the growth and invasive behavior of rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: RASFs were transduced with retroviral constructs expressing dominant-negative mutants of c-Raf-1 or c-Myc (DN c-Raf-1 or DN c-Myc, respectively) or with the mock vector. The expression of wild-type and mutant proteins was confirmed by Western blotting. Growth curves of RASFs were recorded, and apoptosis was measured by flow cytometry. Invasiveness of RASFs was assessed in the SCID mouse model of RA. Immunohistochemistry was used to study the effects of DN c-Raf-1 on phosphorylated c-Jun and matrix metalloproteinase 1 (MMP-1) in RASFs implanted into SCID mice. The phosphorylation of ERK and JNK in DN c-Raf-1- and mock-transduced RASFs was determined in vitro by Western blotting. The levels of MMPs in these cells were measured by quantitative polymerase chain reaction (PCR). RESULTS: Neither DN c-Raf-1 alone nor DN c-Myc alone significantly altered proliferation or apoptosis of RASFs, but both mutants together rapidly induced apoptosis. Inhibition of c-Raf-1 or c-Myc significantly reduced the invasiveness of RASFs in the SCID mouse model. DN c-Raf-1 decreased the phosphorylation of ERK and JNK in vitro and reduced the in vivo expression of phosphorylated c-Jun as well as the expression of disease-relevant MMPs. As determined by quantitative PCR, the inhibition was most pronounced for MMP-1 and MMP-3. CONCLUSION: The data demonstrate that Ras- and c-Myc-dependent signaling events cooperate to regulate the growth and invasiveness of RASFs. Targeting of both c-Raf-1 and c-Myc may constitute an interesting therapeutic approach in RA.

Proinflammatory role of fractalkine (CX3CL1) in rheumatoid arthritis.

OBJECTIVE: Fractalkine (CX3CL1) represents the sole member of the so-called CX3C chemokines. In rheumatoid arthritis (RA), functional studies suggest a role for this chemokine in monocyte chemotaxis and angiogenesis in the rheumatoid synovium. We analyzed the expression of fractalkine within different T cell subsets of the peripheral blood and expression of its receptor CX3CR1 within the rheumatoid synovium to further characterize its pathogenic role in RA. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 17 patients with RA and analyzed by flow cytometry in comparison to healthy blood donors. To identify the T helper cell cytokine profile of fractalkine-expressing cells, flow cytometric analysis of PBMC was performed after stimulation with PMA and ionomycin. Expression of fractalkine and its receptor was characterized in RA synovium by immunohistochemistry and laser capture microdissection microscopy. RESULTS: Flow cytometric analysis of fractalkine-expressing T cell subsets revealed a low proportion of fractalkine-expressing CD4+ and CD8+ T cells in both RA patients and controls. In addition, fractalkine was predominantly expressed in CD4+ T cells with a Th1-type cytokine expression profile. In RA synovium, fractalkine was detected in synovial macrophages, dendritic cells, endothelial cells, and a small proportion of T cells. The fractalkine receptor CX3CR1 was found in synovial macrophages, dendritic cells, and T cells as well as in synovial fibroblasts. Fractalkine stimulation of cultured synovial fibroblasts resulted in a marked upregulation of matrix metalloproteinase-2 (MMP-2) production. CONCLUSION: The results suggest that fractalkine may represent a Th1-type chemokine. Upregulation of MMP-2 production in synovial fibroblasts upon fractalkine stimulation in vitro supports the hypothesis of a proinflammatory role of this chemokine in RA.

Functional characterization of adherent synovial fluid cells in rheumatoid arthritis: destructive potential in vitro and in vivo.

OBJECTIVE: To characterize the morphologic and immunologic features of adherent synovial fluid cells derived from patients with rheumatoid arthritis (RA), and to explore their potential function in vitro and in vivo by focusing on cartilage destruction. METHODS: Synovial fluid adherent cells obtained from patients with RA and from control subjects were characterized by immunohistochemistry, flow cytometry, and electron microscopy. In vitro, these cells were cultured in the presence of cartilage particles. Cartilage destruction was monitored by the release of sulfated glycosaminoglycans (sGAG) into the medium, and the level of matrix metalloproteinase 1 (MMP-1) in the cell culture supernatant was measured by enzyme-linked immunosorbent assay. To inhibit cartilage destruction in vitro, the MMP inhibitor marimastat was tested in this system. In vivo, in the SCID mouse coimplantation model, RA synovial fluid adherent cells and RA synovial fibroblasts (as positive controls) were coimplanted with human cartilage under the kidney capsule and maintained there for 60 days. RESULTS: In vitro, the synovial fluid adherent cells consisted of 2 subpopulations, large round-shaped macrophage-like cells (CD68+) and spindle-shaped fibroblast-like cells (Thy-1+). When passaged, the latter cells proliferated and organized themselves into 3-dimensional formations. This allowed them to reach collagen particles fixed with agarose. Fibroblasts derived from synovial tissues could not be used in this assay because they grew only in monolayers and not on agarose. The majority (>90%) of passaged RA synovial fluid adherent cells expressed the Thy-1+,CD45-,CD68-,CD86- phenotype. Electron microscopy did not reveal important morphologic differences between the 2 types of fibroblasts, those from synovial tissue or those from synovial fluid. However, synovial fluid adherent cells expressed lower levels of adhesion molecules, including CD54 and galectin 3, as well as the complement-regulatory molecule CD55. The in vitro release of sGAG associated with cell activity was 2.5-fold higher from RA synovial fluid adherent cells in comparison with that from negative control cells. The release of sGAG correlated with the concentration of MMP-1 and was inhibited by the broad-range MMP inhibitor marimastat in a dose-dependent manner. RA synovial fluid adherent cells coimplanted with cartilage in SCID mice showed the same invasive behavior as that displayed by tissue-derived RA synovial fibroblasts. CONCLUSION: Similar to tissue-derived RA synovial fibroblasts, RA synovial fluid adherent cells, which contain "floating" anchorage-independent fibroblast-like cells, mediate cartilage destruction independent of the hyperplastic synovial tissue.

Expression of activation-induced, T cell-derived, and chemokine-related cytokine/lymphotactin and its functional role in rheumatoid arthritis.

OBJECTIVE: To evaluate the possible role of activation-induced, T cell-derived, and chemokine-related cytokine (ATAC)/lymphotactin (Lptn) in the pathogenesis of rheumatoid arthritis (RA). METHODS: ATAC/Lptn levels in serum and synovial fluid samples were measured by sandwich enzyme-linked immunosorbent assay. Expression of messenger RNA for ATAC/Lptn in synovial tissues was analyzed by reverse transcription-polymerase chain reaction (PCR) and by in situ hybridization, and was quantitated by real-time PCR. The phenotype of peripheral blood mononuclear cells (PBMCs) expressing ATAC/Lptn was analyzed by intracellular cytokine staining and flow cytometry. RESULTS: Levels of ATAC/Lptn were similar in sera and synovial fluids from RA patients (n = 20) and osteoarthritis controls (n = 15). In phorbol myristate acetate/ionomycin-stimulated PBMCs, ATAC/Lptn expression was detected in CD8+ T cells and in a significantly increased proportion of CD4+,CD28- T cells from RA patients as compared with healthy controls. In synovial tissues, ATAC/Lptn was predominantly localized in CD3+ T cells in the sublining layer. Lymphocytes, synovial macrophages, and, unexpectedly, fibroblast-like synoviocytes (FLS) were identified as major target cells for ATAC/Lptn in RA synovium, as determined by analysis of the ATAC/Lptn receptor XCR1. In vitro, ATAC/Lptn stimulation of FLS resulted in a marked down-regulation of matrix metalloproteinase 2 production. CONCLUSION: These data indicate that in RA synovium, ATAC/Lptn is mainly produced by T cells. Considering its function as a lymphocyte-specific chemoattractant, ATAC/Lptn might be a key modulator for T cell trafficking in the pathogenesis of RA. In addition, functional studies suggest that ATAC/Lptn may exert additional immunomodulatory effects in RA.

Osteoclast-independent bone resorption by fibroblast-like cells.

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.

[Metformin-associated lactic acidosis with acute renal failure in type 2 diabetes mellitus]. / Metformin-assoziierte Lactatazidose mit akutem Nierenversagen bei Diabetes mellitus Typ 2.

CASE REPORT: An 83-year-old patient was admitted to our hospital because of gastrointestinal symptoms, mental confusion and dysarthria. The patient suffered from type 2 diabetes mellitus and was taking metformin. A mild renal insufficiency was known. On admission, we found impaired consciousness, Kussmaul breathing, a body temperature of 32.1 degrees C, and hemodynamic instability. Laboratory testing revealed lactic acidosis (pH 6.71, base excess--30, standard bicarbonate 4.0 mmol/l, lactate 24.4 mmol/l) and acute renal failure with a creatinine of 10.6 mg/dl and blood urea nitrogen of 134 mg/dl. Electrolytes were not altered; the blood glucose was elevated (147 mg/dl). According to history, physical examination, and laboratory testing the diagnosis metformin-induced lactic acidosis with acute renal failure was made. This diagnosis was confirmed by an elevated level of metformin. As soon as possible a bicarbonate hemodialysis was initiated. After 8 hours of hemodialysis the acid-base metabolism was almost balanced and the vigilance of the patient normalized. No further sessions of hemodialysis were needed and insulin therapy was started. CONCLUSIONS: Metformin-induced lactic acidosis is a common side effect in patients with renal insufficiency. For an early diagnosis, clinical symptoms of intoxication should be well known by physicians and patients. First-line therapy for correction of lactic acidosis and effective elimination of metformin is bicarbonate hemodialysis. Sodium bicarbonate infusions are not able to correct the acid-base metabolism sufficiently. For prevention the renal function should be monitored closely and metaformin therapy should be stopped, if a deterioration of renal function is observed.